Proteins are involved in all physiological processes, from cell signaling to tissue remodeling to organ function. Thus, protein synthesis in cells is a very important process, and ribosomes perform this function. Ribosomes are cell organelles that connect amino acids to form proteins, and are large subunits composed of ribosomal proteins and ribosomal RNA.
rpS3 (ribosomal protein S3), a component of ribosomes, is located on the outer surface of the 40S subunit and is cross-linked to the initiation factor eIF2 and eIF3. As rpS3 has a nuclear localization of signal in the N-terminal region, it is believed that the translation functions of rpS3 operate in the cytosol, and the repair function operates in the nucleus. In addition to their role in ribosomal functions, many ribosomal proteins have secondary functions in replication, transcription, RNA processing, DNA repair, and malignant transformation.
The rpS3 gene is located on human chromosome 11q13.3-q13.5. Particularly, it was reported that the rpS3 gene is overexpressed in colorectal cancer patients. Thus, rpS3 gene products are overexpressed in colorectal cancer, and are highly likely to be associated with other cancers. In addition, studies on the region 11q13.3-q13.5 indicate that the structural abnormality and amplification of the gene often occur, and the gene is overexpressed upon the development of human cancers such as multiple endocrine neoplasia type I, breast carcinoma or B cell neoplasia (Pogue-Geile et al., Mol. Cell. Biol., 11: 3842-3849, 1991).
As used herein, the term “siRNA (small interference RNA)” refers to a short double-strand RNA composed of about ten nucleotides to several tens of nucleotides, which induce RNAi (RNA interference). RNA interference (“RNAi”) is a method of post-transcriptional inhibition of gene expression that is conserved throughout many eukaryotic organisms, and it refers to a phenomenon in which a double-stranded RNA composed of a sense RNA having a sequence homologous to the mRNA of the target gene and an antisense RNA having a sequence complementary thereto is introduced into cells or the like so that it can selectively induce the degradation of the mRNA of the target gene or can inhibit the expression of the target gene. RNAi is induced by a short (i.e., less than 30 nucleotides) double-stranded RNA (“dsRNA”) molecule present in cells (Fire A. et al., Nature, 391: 806-811, 1998). Because RNAi can selectively inhibit the expression of the target gene as described above, it is attracting considerable attention as a simple gene knock-down method that acts as a substitute for a conventional gene destruction method based on inefficient homologous recombination. When siRNA is introduced into cells, the expression of the mRNA of the target gene having a nucleotide sequence complementary to that of the siRNA will be inhibited.]
The siRNA and the targeted mRNA bind to an RNA-induced silencing complex (“RISC”), which cleaves the targeted mRNA. The siRNA is apparently recycled much like a multiple-turnover enzyme, with 1 siRNA molecule capable of inducing cleavage of approximately 1000 mRNA molecules. Thus, siRNA-mediated RNAi degradation of an mRNA is more effective than currently available technologies for inhibiting expression of a target gene.
However, unlike the initial expectation that RNAi technology can be applied to the RNA of all genes to selectively inhibit the expression of the RNA, it has recently been found that the RNAi technology is not applied, because some genes are unknown in high-throughput RNAi screening procedures (Krueger et al., Oligonucleotides, 17:237-250, 2007). In addition, in past several years, algorithms for predicting siRNA sequences by statistical analysis based on databases have been developed. However, it is known that, because computational algorithms are not perfect, all siRNAs determined by an in silico method using the algorithm do not effectively inhibit the target mRNA in cells and in vivo, and the most efficient siRNA can be determined only by experimental verification (Derek et al., Annu. Rev. Biomed. Eng., 8:377-402, 2006).
As described above, theoretically predicted siRNAs must not effectively inhibit the ability of mRNA, and highly efficient siRNA nucleotide sequences should be found to achieve therapeutic purposes. For this reason, it is very important to find a specific hyperactive target having high inhibitory effects on the target mRNA. In addition, it is required to identify the optimum sequence having an excellent expression inhibitory effect by preparing siRNAs based on a number of target sites selected per mRNA of a single gene and directly examining the effects of the siRNAs directly, at least at the cellular level.
Accordingly, the present inventors have made extensive efforts to develop an siRNA for inhibiting the expression of ribosomal protein S3 (rpS3), and as a result, have constructed an rpS3 siRNA that bind to the specific target site of rpS3, and have found that the constructed siRNA has an excellent effect of inhibiting the expression of endogenous rpS3 protein, compared to commercially available products, thereby completing the present invention.